An enzyme-powered, three-dimensional lame DNA walker

Abstract Molecular machines constructed by three-dimensional (3-D) DNA walker have emerged as a hot topic in applications such as novel biosensors, cargo delivery platforms and intracellular imaging. Herein, we first propose a lame DNA walker that can randomly and autonomously move on microsphere-based 3-D track. The stochastic lame walker has a long leg mainly responsible for persistent movement and a short leg cutting substrates rapidly. Its motion is propelled by a nicking endonuclease cleavage of hybridized DNA tracks. Kinetic and persistent study show that the lame DNA walker enables reaction equilibrium at 30 min, need a cleat domain of at least 14 nucleotides and can persistently move on 3-D tracks with an average rate of 6.467 × 10−11 M s−1. We also demonstrate that the lame walker can be used to detect target DNA in the detection range of 10 pM–5 nM with high specificity by toehold exchange mechanism. This work will further expand the performance of 3-D DNA walkers and substantially contributes to the improved understanding of DNA walking systems.